Rizwana Quraishi

Use of filter paper stored dried blood for measurement of triglycerides

Adaptation of assays on dried blood has advantages of ease of collection, transportation, minimal invasiveness and requirement of small volume. A method for extraction and estimation of triglyceride from blood spots dried on filter paper (Whatman no. 3) has been developed. A single dried blood spot containing 10 μL blood was used. Triglyceride was efficiently extracted in methanol from blood dried on filter paper by incubation at 37°C for two hours with gentle shaking. For the estimation, a commercially available enzymatic method was used. Blood spot assays showed mean intra and inter assay coefficient of variance of 6.0% and 7.4% respectively. A comparison of paired whole blood spots and plasma samples (n = 75, day 0) gave an intraclass correlation of 0.96. The recovery was 99.6%. The dried blood triglyceride concentrations were stable for one month when the filter discs were stored at room temperature (16–28°C). Storage of filters at 4°C extended the stability and triglycerides could be quantatively recovered after 3 months of storage.

Comparison of self-report and biological measures for alcohol, tobacco and illicit drug use in consecutive alcohol-dependent patients visiting a tertiary care centre

Background: Alcohol is a major drug of abuse in patients visiting treatment centres. The self-report is used by clinicians to collect information; however, alcohol-dependent patients may not provide reliable self-reports. Aim: The study aimed to assess the agreement between urinalysis and self-report for licit and illicit drug use in alcohol-dependent patients and to examine the relationship of biochemical measures with self-reported alcohol consumption. Methods: Consecutive patients (N = 199) with current alcohol use fulfilling Diagnostic and Statistical Manual, 4th edition (DSM-IV) criteria for alcohol dependence were included. Recent alcohol and drug use was enquired using a structured questionnaire. The biological measures included urine drug analysis and biochemical liver function tests. Results: Urinalysis revealed the use of opiates, cannabis, nicotine and benzodiazepines in 2.5%, 4.2%, 5.9% and 28.8%, respectively, even though patients reported no use of these drugs. Gamma-glutamyl transferase (GGT) was elevated in 95.2% of patients with self-reported heavy alcohol consumption, whereas it was raised in 60–62.5% of those with low to moderate alcohol use. In heavy drinkers, the biochemical liver markers appeared to corroborate with self-reported alcohol use. Conclusion: This study urges for the use of objective methods to validate the self-report in alcohol-dependent patients, especially for co-occurring nonprescription benzodiazepine and drug use.

Profile of nicotine use among alcohol dependent patients visiting a tertiary care center in north India

Background: Use of tobacco among alcohol dependent population is quite frequent. This co-morbidity increases the risk for various diseases. Understanding the pattern of tobacco use with co-morbid alcohol use may help in planning appropriate prevention/treatment strategies. The study aimed at examining the profile and pattern of nicotine use among alcohol dependent patients visiting a tertiary care treatment center in North India. Materials and Methods: Male patients fulfilling diagnostics and statistical manual of mental disorder fourth edition, criteria for nicotine and alcohol diagnostics and statistical dependence, attending the out-patient department of the tertiary care treatment center were recruited after obtaining informed consent. The socio-demographic profile, drug use history, nicotine associated health problems and general health problem were recorded. Motivation to stop tobacco use was assessed qualitatively using the direct questions about their interest and intentions to quit. Results: A total of 150 subjects were included in the study. The mean age of the study sample was 37.6 ± 10.44 years. Tobacco was reported as the gateway drug in 90% of the cases. Exclusive bidi use reported in 42% of the subjects. Mean duration of bidi and co-morbid alcohol use was higher than cigarette or smokeless tobacco use. Self-reported health problems associated with nicotine use and general health was reported by 41% and 39% of the subjects. Unsuccessful past quit attempts was present in 85% cases. More than 90% of subjects remained interested in quitting the tobacco use. An increased liver enzyme (aspartate transaminase, alanine transaminase and gamma-glutamyl transferase) were observed in 43, 32 and 47% of the cases. Conclusion: The results suggest the nicotine and alcohol dependent patients represent a separate population requiring higher attention from the treating physician.

A descriptive study of clinical, hematological, and biochemical parameters of inhalant users seeking treatment at a tertiary care center in India

Background: Inhalant (or solvent) abuse is the purposeful inhalation of vapors or gases, intended to produce pleasurable psychoactive effects. There is a dearth of Indian studies on inhalant users. Aim: The present study aimed to describe the socio-demographic, clinical, and psychosocial characteristics of inhalant users visiting a Tertiary Care Center in North India. Materials and Methods: The study was a retrospective chart review for 50 inhalant users who sought treatment for the first time from the center over a period of 2 years. All patients seeking treatment for inhalant use at the center were evaluated by a psychiatrist. Results: Mean age of the sample was 17.16±4.09 years and majority comprised of children and adolescents (72.2%). There were only three girls (6%). Majority comprised of school drop-outs (82%), from lower socio-economic status (80%). Mean age of initiation of first substance was 14.13±4.27 years and inhalants were first drugs for 38%. Duration of inhalant use ranged between 1 month and 7.5 years. Use was mostly uninterrupted, and 88% were dependent users. Correction fluid was the commonest product, used by huffing or sniffing. A large majority (86%) had used at least one other substance besides inhalants, and 8% reported involvement in high-risk sexual behaviors. Comorbid psychiatric disorder was seen in 8% of sample. Positive family history was observed in 30% of the sample. The mean hemoglobin of the sample was 11.88±0.60, with low hemoglobin in 25% of users. Neutrophils, lymphocytes, eosinophils, and monocytes were elevated beyond normal in 10.8%, 6.5%, 15.2%, and 7.5%, respectively. There was no evidence of leucopenia. Bilirubin and serum glutamic pyruvic transaminase was elevated in 6.6% and 13% of inhalant users, respectively. Conclusion: The study adds to the limited date available on the treatment-seeking inhalant users from Indian settings. There is a need to examine the pattern of inhalant use in larger samples, across multiple sites in a prospective manner.

Creatinine measurement and stability in dried serum.

Studies have shown an association between serum creatinine levels and type 2 diabetes in various populations.1–2 For epidemiological studies and screening programs, use of a filter paper matrix circumvents transportation issues and has been reported for many analyses.3–6 However, the use of dried blood or serum has not been reported for the measurement of creatinine. This study reported the use of dried serum for the measurement of creatinine. For the study, 60 leftover serum samples from the subjects’ visiting department of laboratory medicine, All India Institute of Medical Sciences, were collected for laboratory investigations. Ethical clearance for the conduct of the study was obtained from institutional ethics committee. An aliquot of each serum sample was analyzed immediately. Remaining aliquot was spotted on filter paper. Multiple spots were approximately 10 μl, and each was prepared on 3 micron Whatman™ filter paper (Whatman plc., Kent, United Kingdom). After drying, the discs were kept in sealed plastic bags and stored at 37 ˚C and at 4 ˚C. For serum creatinine estimation, 100 μl of the serum was added to 1 ml of the colorimetric reagent (Randox Laboratories Ltd., Crumlin, United Kingdom). For dried plasma creatinine estimation, five (6 mm) discs were punched from the prepared spots and dropped into 10 ml glass tubes containing 1 ml of the colorimetric reagent. For fresh serum, the reaction time was 10 min. The extraction from dried serum was carried out for a period of 30 min, with gentle shaking at 100 rates per min in an environ shaker (Lab-Line Instruments Inc., Melrose Park, IL) at 37 oC. The final absorbance was read at 540 nm wavelength. Calibration was done with calibrators spotted onto filter paper and treated the same way as samples. Total creatinine values in the 60 samples (fresh) ranged from 0.5 to 3.3 mg/dl. The creatinine levels obtained in serum samples were compared with paired dried serum samples on the day of collection. Creatinine mean (± standard deviation) values in serum and their dried serum values were 1.99 (± 0.64) and 1.92 (± 0.55) mg/dl, respectively. The values obtained by the two methods correlate well with the r value of 0.94 and intraclass correlation value of 0.93 with 96% recovery on the day of sample collection. To assess stability of the dried serum samples, discs were punched from a subsample size (n = 15), and creatinine levels were estimated at the end of 0, 15, 30, 60, and 90 days. Mean creatinine value in dried serum eluted and measured on the day of collection (day 0) did not differ significantly from other storage days. Storage of filter disc at 37 ˚C and at 4 ˚C for 15, 30, 60, and 90 days did not significantly alter the mean creatinine values (Table 1). Table 1 Effect of Storage of Dried Serum on Creatinine Concentrations This study demonstrates filter paper as a feasible matrix for collection and transport of serum for creatinine measurement. This approach needs to be evaluated in whole blood also. The stability of creatinine in a filter matrix for up to 3 months makes this method very promising for epidemiological studies. We conclude that creatinine is highly stable in dried serum and is readily transferable to a liquid phase for epidemiological studies.

Laboratory Profiles of Treatment-Seeking Subjects With Concurrent Dependence on Cannabis and Other Substances: A Comparative Study

Background: Cannabis is one of the most widely used illicit drugs in India and worldwide. It is considered to have a minimal effect on physical health. Objectives: The aim of this study was to compare the laboratory profiles of treatment-seeking patients who were cannabis dependent, and drug users who concurrently use other substances, with non-users. Materials and Methods: Medical records of patients, whose urine was tested for the detection of cannabis within the last year, were considered for the study. The inclusion criteria for the study group were; co-morbid diagnosis of cannabis dependence according to DSM-IV TR criteria, positive urine drug screen for cannabis, and at least one biochemical or hematological examination report during the treatment period. The subjects who underwent all of the above mentioned tests, but who were negative for any psychoactive substance with no past or current history of substance use, were placed in the control group. Results: A total of 51 subjects fulfilled the inclusion criteria for the study group and 30 subjects were considered as controls. There was no significant difference found between the demographic profiles of the subject and control groups. The mean duration of cannabis use in the patients was 9.53 ± 8.06 years. Serum levels of; bilirubin, SGOT (serum glutamic oxaloacetic transaminase), SGPT (serum glutamic pyruvic transaminase), total protein, alkaline phosphatase, ESR, and eosinophil counts, were raised in; 13.7%, 15.6%, 33.3%, 17.6%, 37.2%, 75% and 5.8% of subjects, respectively. The relative monocyte count was lower than normal in 92% of cases. Physical complaints were reported in 98% of subjects. The two groups showed significant differences in serum alkaline phosphatase [t (79) = 6.5, P ≤ 0.01], TLC [t (79) = 2.36, P = 0.03] and hemoglobin levels [t (79) = 5.50, P ≤ 0.01]. Conclusions: Abnormal laboratory parameters were observed in patients with cannabis dependence. The study emphasizes the need for regular physical examinations and laboratory investigations for cannabis users.

Evaluation of Dried Urine Spot Method to Screen Cotinine among Tobacco Dependents: An Exploratory Study

Background and Objectives: Assessment of cotinine, a metabolite of nicotine in body fluids, is an important approach for validating the self-report among tobacco users. Adaptation of assays on dried urine spots (DUSs) has advantages of ease of collection, transportation, minimal invasiveness, and requirement of small volume. The aim of the present study was to develop an efficient method for testing cotinine in DUSs and evaluating its clinical applicability. Methods: This involved optimization of conditions for detection, recovery, and stability of cotinine from dried urine, spotted on filter paper. Enzyme-linked immunosorbent assay was used for screening, whereas confirmation was done by gas chromatography. For clinical applicability, urine samples of tobacco users were tested. Results and Interpretation: Water was found to be a suitable extracting solvent as compared to carbonate-bicarbonate buffer (pH 9.2) and saline. Screening was achieved by two punches taken from a 20 μl (diameter 1.3 cm) spotted urine samples, and confirmation was achieved by five complete circles each of 20 μl sample volume. The recovery was found to be 97% in water. Limit of detection for the method was found to be 100 ng/ml. No signs of significant degradation were found under all storage conditions. All the urine samples of tobacco users were found to be positive by a conventional method as well as DUSs, and the method proved to be efficient. Conclusions: DUS samples are a useful alternative for biological monitoring of recent nicotine use, especially in developing countries where sample logistics could be an important concern.

Association of ankyrin repeats & kinase domain containing 1 (ANKK1) gene polymorphism with co-morbid alcohol & nicotine dependence: A pilot study from a tertiary care treatment centre in north India

Background & objectives: The frequently encountered co-morbidity of alcohol dependence (AD) with nicotine dependence (ND) increases the risk for various diseases. Ankyrin repeats and kinase domain containing 1 (ANKK1) gene polymorphism is reported to be associated with both ND and AD. This study was undertaken to investigate the possible association of alcohol and tobacco use variables with ANKK1 polymorphism in co-morbid alcohol- and nicotine-dependent treatment seekers visiting a tertiary care centre in north India. Methods: Seventy nine male participants (18-65 yr old) fulfilling diagnostic criteria for ND and AD were included in the study. The socio-demographic data, along with alcohol and tobacco use profile, was recorded and ANKK1 profiling was carried out. Both the allele groups, A1 and A2, were compared with respect to demographic and substance dependence profile. Univariate binary logistic regression analysis was performed to determine the risk of high nicotine and alcohol consumption with genotype. Results: The A1 carrier group (n=33) reported a significantly higher amount of alcohol and tobacco consumed per day. The scores on parameters of ND were found to be significantly higher in this group. The logistic regression analysis revealed that participants with A1 genotype were 2.5 times more likely to report higher amount of alcohol and nicotine consumption than A2 carriers. Interpretation & conclusions: The study provides an indication for the association of ANKK1 polymorphism in the form of higher substance consumption among alcohol dependent smokers, who are A1 carriers and thus may require higher attention of the treatment provider.

Sociodemographic and Drug Use Characteristics of Treatment-seeking Cannabis Users at a Tertiary Care Center in India

Background:Cannabis is a common drug of abuse that has got some religious and social sanction in India. It is often perceived as relatively harmless compared with other substances. The rate of treatment seeking is low among its users. Objective:To describe the sociodemographic and drug use profile of treatment-seeking cannabis users visiting a tertiary care center in northern India. Methods:A retrospective chart review was conducted over a period of 1 year for cannabis-dependent subjects who sought treatment and underwent laboratory investigations. Results:A total of 51 subjects fulfilled the DSM-IV-TR criteria for cannabis dependence. All subjects were male with mean age of 28±10.2 years. Among them, 37.3% subjects started substance use before 15 years of age. Although 94% subjects were literate, only 33.3% were employed and 82% were socioeconomically poor. Majority of the subjects initiated substance use with smokable tobacco (66.7%). Cannabis was the initial substance among 15% subjects. Among all 64.7% subjects were comorbid opioid dependent and 58.9% subjects were seeking treatment for the same. Ganja was the most common form of cannabis used (66.7%). Health risk due to cannabis was known to 35.3% subjects, 47% never made any abstinent attempt. Cannabis-related complication was present in 84.37% subjects. Conclusions:Treatment-seeking cannabis users are mostly codependent on other substances that become the main reason for their treatment-seeking. Despite high rate of cannabis-related complications, awareness about its health-risk, treatment seeking, and attempt to abstain remains low.

Naltrexone Level and Biochemical Profile of Opiate-dependent Patients on Naltrexone Maintenance Program: A Pilot Study

Objectives:Naltrexone is a potent narcotics antagonist, but its utility is limited by the patient’s noncompliance. The present study was conducted to ascertain the naltrexone levels in patients maintained on daily dose of naltrexone along with their biochemical profile. Methods:Patients, fulfilling DSM (IV) criteria, who were on naltrexone maintenance program were included. Two hours after the intake of 50 mg of naltrexone, 5 mL of the blood and urine samples were collected from each patient. Plasma was separated for naltrexone estimation and urinalysis was performed to ascertain illicit opioid use. The biological markers and sociodemographic profile were also recorded. Results:The study included 10 male patients. The median age of patients was 28.5 years. The duration of drug use varied from 6 months to 27 years, with a median age of 8 years of opioid use. One patient reported high-risk sexual behavior. The biochemical profile was within the normal range except for 1 patient. The plasma concentration of naltrexone ranged from 0.12 to 0.70 &mgr;g/mL with a mean value of 0.49 &mgr;g/mL. Conclusions:These results suggest that plasma naltrexone levels along with the biochemical profile can be used as a reliable measure for assessment of compliance of antagonist therapy in opioid-dependent patients in Indian population. The findings need to be replicated with a bigger sample size.